Project Summary

This project aimed to look at the spatial variability of Symbiodinium clades C and D in the Kane’ohe Bay, O’ahu, Hawai’i population of Montipora capitata. We investigated the distribution of symbionts at scales ranging from location in the bay to location on an individual reef. We also looked at differences among reef types (fringing vs. patch), colony color morph (brown vs. orange) and depth. Heterogeneous mixtures of symbiont clades were considered in the analysis for spatial patterns. By investigating spatial variability of Symbiodinium, we furthered the understanding of stress-response potential in Kane’ohe Bay.

Library Packages

setwd("~/Symcap")
library(data.table)
library(devtools)
library(plyr)
library(reshape2)
library(popbio)
library(RgoogleMaps)
library(plotrix)
library(zoo)
library(rgdal)
library(car)
library(scales)
library(png)
library(pixmap)
library(ecodist)

Import/Merge Field and qPCR Data

Coral_Data <- read.csv("Coral_Collection.csv")
Coral_Data$Depth..m. <- as.numeric(as.character(Coral_Data$Depth..m.))
source_url("https://raw.githubusercontent.com/jrcunning/steponeR/master/steponeR.R")
Mcap.plates <- list.files(path = "qPCR_data", pattern = "txt$", full.names = T)
Mcap <- steponeR(files = Mcap.plates, delim="\t",
                 target.ratios=c("C.D"), 
                 fluor.norm = list(C=2.26827, D=0), 
                 copy.number=list(C=33, D=3))
Mcap <- Mcap$result
Mcap <- Mcap[grep("+", Mcap$Sample.Name, fixed=T, invert = T), ]
Mcap <- Mcap[grep("NTC", Mcap$Sample.Name, fixed = T, invert = T), ]
Mcap <- Mcap[grep("PCT", Mcap$Sample.Name, fixed = T, invert = T), ]
colnames(Mcap)[which(colnames(Mcap)=="Sample.Name")] <- "Colony"
Mcap$fail <- ifelse(Mcap$C.reps < 2 & Mcap$D.reps < 2, TRUE, FALSE)
fails <- Mcap[Mcap$fail==TRUE, ]
Mcap <- Mcap[which(Mcap$fail==FALSE),]
Mcap$C.D[which(Mcap$C.reps<2)] <- -Inf
Mcap$C.D[which(Mcap$D.reps<2)] <- Inf
Mcap <- Mcap[with(Mcap, order(Colony)), ]
Mcap$propC <- Mcap$C.D / (Mcap$C.D+1)
Mcap$propD <- 1-Mcap$propC
Mcap$propD[which(Mcap$C.D==-Inf)] <-1
Mcap$propC[which(Mcap$C.D==-Inf)] <-0
Mcap$propD[which(Mcap$C.D==Inf)] <-0
Mcap$propC[which(Mcap$C.D==Inf)] <-1
Mcap$Dom <- ifelse(Mcap$propC>Mcap$propD, "C", "D")
Symcap<-merge(Coral_Data, Mcap, by="Colony", all=T)
Symcap <- Symcap[with(Symcap, order(Colony)), ]
Symcap$Mix <- factor(ifelse(Symcap$propC>Symcap$propD, ifelse(Symcap$propD!=0, "CD", "C"), ifelse(Symcap$propD>Symcap$propC, ifelse(Symcap$propC!=0, "DC", "D"), NA)), levels = c("C", "CD", "DC", "D"))
Symcap$Reef.Area <- ifelse(Symcap$Reef.Area!="Top", yes = "Slope", no = "Top")

Adjust Depth by Mean Sea Level

To account for the influence of tides, depth was adjusted according to the difference in sea level from the mean sea level on each collection day at 6-minute intervals. Mean sea level was obtained from NOAA tide tables for Moku o Lo’e.

JuneTide=read.csv("Station_1612480_tide_ht_20160601-20160630.csv")
JulyTide=read.csv("Station_1612480_tide_ht_20160701-20160731.csv")
AugustTide=read.csv("Station_1612480_tide_ht_20160801-20160812.csv")
Tide<-rbind(JuneTide, JulyTide, AugustTide)
Tide$Time <- as.POSIXct(Tide$TimeUTC, format="%Y-%m-%d %H:%M:%S", tz="UTC")
attributes(Tide$Time)$tzone <- "Pacific/Honolulu"
Symcap$Time2 <- as.POSIXct(paste(as.character(Symcap$Date), as.character(Symcap$Time)),
                                format="%m/%d/%y %H:%M", tz="Pacific/Honolulu")
Symcap$Time=Symcap$Time2

# Add estimated times for missing values
Symcap$Time[170] <- as.POSIXct("2016-06-14 12:07:00")
Symcap$Time[177] <- as.POSIXct("2016-06-14 12:20:00")
Symcap$Time[178] <- as.POSIXct("2016-06-14 12:22:00")
Symcap$Time[180] <- as.POSIXct("2016-06-14 13:08:00")
Symcap$Time[187] <- as.POSIXct("2016-06-14 12:42:00")
Symcap$Time[188] <- as.POSIXct("2016-06-14 12:44:00")
Symcap$Time[206] <- as.POSIXct("2016-06-16 13:10:00")
Symcap$Time[208] <- as.POSIXct("2016-06-16 13:24:00")
Symcap$Time[211] <- as.POSIXct("2016-06-16 12:37:00")
Symcap$Time[218] <- as.POSIXct("2016-06-16 12:27:00")
Symcap$Time[448] <- as.POSIXct("2016-07-16 13:32:00")

Round6 <- function (times)  {
  x <- as.POSIXlt(times)
  mins <- x$min
  mult <- mins %/% 6
  remain <- mins %% 6
  if(remain > 3L) {
    mult <- mult + 1
  } else {
    x$min <- 6 * mult
  }
  x <- as.POSIXct(x)
  x
}

Symcap$Time.r <- Round6(Symcap$Time)
Tide$Time.r <- Tide$Time
merged<-merge(Symcap, Tide, by="Time.r", all.x=T)
merged$newDepth <- merged$Depth..m.- merged$TideHT

Chi-Squared

Significant Differences

Dominant Symbiont by Reef Area

Symcap$Reef.Area <- ifelse(Symcap$Reef.Area!="Top", yes = "Slope", no = "Top")
results=table(Symcap$Dom, Symcap$Reef.Area)
chisq.test(results)
## 
##  Pearson's Chi-squared test with Yates' continuity correction
## 
## data:  results
## X-squared = 136.26, df = 1, p-value < 2.2e-16
prop.table(results, margin = 2)
##    
##         Slope       Top
##   C 0.7767857 0.3294574
##   D 0.2232143 0.6705426
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray10", "gray100"), xlab = "Reef Area", ylab = "Symbmiont Proportion")
legend("topright", legend=c("C", "D"), fill=c("gray10", "gray100"), inset = c(-.2, 0), xpd = NA)

Dominant Symbiont by Color Morph

results=table(Symcap$Dom, Symcap$Color.Morph)
chisq.test(results)
## 
##  Pearson's Chi-squared test with Yates' continuity correction
## 
## data:  results
## X-squared = 164.96, df = 1, p-value < 2.2e-16
prop.table(results, margin = 2)
##    
##         Brown    Orange
##   C 0.8896321 0.4103194
##   D 0.1103679 0.5896806
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray10", "gray100"), xlab = "Color Morph", ylab = "Symbiont Proportion")
legend("topright", legend=c("C", "D"), fill=c("gray10", "gray100"), inset = c(-.2, 0), xpd = NA)

Symbiont Community Composition by Color Morph

results=table(Symcap$Mix, Symcap$Color.Morph)
chisq.test(results)
## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 167.44, df = 3, p-value < 2.2e-16
prop.table(results, margin = 2)
##     
##            Brown      Orange
##   C  0.762541806 0.361179361
##   CD 0.127090301 0.049140049
##   DC 0.107023411 0.570024570
##   D  0.003344482 0.019656020
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray10", "gray50", "gray85", "gray100"), xlab = "Color Morph", ylab = "Symbiont Community Composition")
legend("topright", legend=c("C", "CD", "DC", "D"), fill=c("gray10", "gray50", "gray85", "gray100"), inset = c(-.2, 0), xpd = NA)

Symbiont Community Composition by Reef Area

Symcap$Reef.Area <- ifelse(Symcap$Reef.Area!="Top", yes = "Slope", no = "Top")
results=table(Symcap$Mix, Symcap$Reef.Area)
chisq.test(results)
## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 138.97, df = 3, p-value < 2.2e-16
prop.table(results, margin = 2)
##     
##            Slope         Top
##   C  0.678571429 0.275193798
##   CD 0.098214286 0.054263566
##   DC 0.214285714 0.651162791
##   D  0.008928571 0.019379845
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray10", "gray50", "gray85", "gray100"), xlab = "Reef Area", ylab = "Symbiont Community Composition")
legend("topright", legend=c("C", "CD", "DC", "D"), fill=c("gray10", "gray50", "gray85", "gray100"), inset = c(-.2, 0), xpd = NA)

Symbiont Community Composition by Dominant Symbiont

results=table(Symcap$Mix, Symcap$Dom)
chisq.test(results)
## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 707, df = 3, p-value < 2.2e-16
prop.table(results, margin = 2)
##     
##               C          D
##   C  0.86635945 0.00000000
##   CD 0.13364055 0.00000000
##   DC 0.00000000 0.96703297
##   D  0.00000000 0.03296703
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray 10", "gray 85", "gray 40", "gray100"), xlab = "Dominant Symbiont", ylab = "Symbiont Mixture Proportion")
legend("topright", legend=c("C", "CD", "DC", "D"), fill=c("gray10", "gray85", "gray40", "gray100"), inset = c(-.2, 0), xpd = NA)

When looking at D-only colonies, the Ct values are on the higher end of the spectrum (35 or greater) indicating poor amplification and the potential for C amplification being pushed back in the cycle. This is supported by the fact that 5 of the 9 D-only colonies had C present in 1 qPCR replicate.

Proportion of D When Present in Mixture

propD <- merged$propD[which(merged$propD > 0 & merged$propD < 1)]
hist(propD, xlab = "Proportion of Clade D", ylab = "Number of Samples", main = "", col = "gray75")

propDHist <- subset(merged, propD > 0 & propD < 1)
propDHist$propD <- cut(propDHist$propD, breaks = 10)
DCol=table(propDHist$Color.Morph, propDHist$propD)
par(mar=c(2, 4, 2, 6))
barplot(DCol, xlab = "Clade D Proportion", ylab = "Number of Samples", 
        main = "", col = c(alpha("sienna", 0.55), alpha("orange", 0.55)), 
        axisnames = FALSE, space = 0)

par(new = T, mar = c(8, 5, 1, 15))
img <- readPNG("brown_orange.png")
img2 <- pixmapRGB(img)
plot(img2)

Color Morph by Reef Area

Symcap$Reef.Area <- ifelse(Symcap$Reef.Area!="Top", yes = "Slope", no = "Top")
results=table(Symcap$Color.Morph, Symcap$Reef.Area)
chisq.test(results)
## 
##  Pearson's Chi-squared test with Yates' continuity correction
## 
## data:  results
## X-squared = 81.109, df = 1, p-value < 2.2e-16
prop.table(results, margin = 2)
##         
##              Slope       Top
##   Brown  0.5523385 0.2015504
##   Orange 0.4476615 0.7984496
par(mar=c(4, 4, 2, 6))
barplot(prop.table(results, margin = 2), col = c("gray10", "gray100"), xlab = "Reef Area", ylab = "Color Morph Proportion")
legend("topright", legend=c("Brown", "Orange"), fill=c("gray10", "gray100"), inset = c(-.2, 0), xpd = NA)

Color Morph by Reef ID

results=table(Symcap$Color.Morph, Symcap$Reef.ID)
chisq.test(results)
## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 50.691, df = 24, p-value = 0.001156
prop.table(results, margin = 2)
##         
##                 10        13        14        18        19        20
##   Brown  0.4000000 0.4000000 0.5333333 0.2333333 0.3666667 0.6285714
##   Orange 0.6000000 0.6000000 0.4666667 0.7666667 0.6333333 0.3714286
##         
##                 21        25        26        30        38        42
##   Brown  0.4333333 0.6000000 0.4242424 0.1333333 0.2857143 0.5142857
##   Orange 0.5666667 0.4000000 0.5757576 0.8666667 0.7142857 0.4857143
##         
##                 46         5      Deep     F1-46     F2-25     F3-18
##   Brown  0.5000000 0.3666667 0.5172414 0.4000000 0.6000000 0.2500000
##   Orange 0.5000000 0.6333333 0.4827586 0.6000000 0.4000000 0.7500000
##         
##              F4-34     F5-34 F6-Lilipuna     F7-18     F8-10      F9-5
##   Brown  0.4400000 0.2500000   0.5000000 0.2000000 0.4000000 0.4000000
##   Orange 0.5600000 0.7500000   0.5000000 0.8000000 0.6000000 0.6000000
##         
##               HIMB
##   Brown  0.6000000
##   Orange 0.4000000
KB <- c(21.46087401, -157.809907) 
KBMap <- GetMap(center = KB, zoom = 13, maptype = "satellite", SCALE = 2, GRAYSCALE = FALSE)
Latitude=aggregate(Latitude~Reef.ID, data=Symcap, FUN = mean)
Longitude=aggregate(Longitude~Reef.ID, data = Symcap, FUN=mean)
XY<-merge(Latitude, Longitude, by="Reef.ID", all=T)
propCol=table(Symcap$Color.Morph, Symcap$Reef.ID)
propCol=prop.table(propCol, margin = 2)
propCol <- as.data.frame.matrix(propCol)
props <- data.frame(t(propCol))
props$Reef.ID <- rownames(props)
XY<-merge(XY, props, by="Reef.ID", all=T)
newcoords <- LatLon2XY.centered(KBMap, XY$Latitude, XY$Longitude, zoom=13)
XY$X <- newcoords$newX
XY$Y <- newcoords$newY
PlotOnStaticMap(KBMap, XY$Latitude, XY$Longitude)

rownames(XY) <- XY$Reef.ID
XY <- XY[, -1]
XY <- na.omit(XY)
apply(XY, MARGIN=1, FUN=function(reef) {
  floating.pie(xpos = reef["X"], ypos = reef["Y"], 
               x=c(reef["Orange"], reef["Brown"]), radius = 7, col = c("orange", "sienna"))
})

Mantel Test for Spatial Autocorrelation

Latitude=aggregate(Latitude~Reef.ID, data=Symcap, FUN = mean)
Longitude=aggregate(Longitude~Reef.ID, data = Symcap, FUN=mean)
XY<-merge(Latitude, Longitude, by="Reef.ID", all=T)
propCol=table(Symcap$Color.Morph, Symcap$Reef.ID)
propCol=prop.table(propCol, margin = 2)
propCol <- as.data.frame.matrix(propCol)
props <- data.frame(t(propCol))
props$Reef.ID <- rownames(props)
XY<-merge(XY, props, by="Reef.ID", all=T)
XY$X <- newcoords$newX
XY$Y <- newcoords$newY
reef.dists <- dist(cbind(XY$Longitude, XY$Latitude))
col.dists <- dist(XY$Brown)
mantel(col.dists~reef.dists)
##     mantelr       pval1       pval2       pval3   llim.2.5%  ulim.97.5% 
## -0.09132955  0.88900000  0.11200000  0.24800000 -0.15070534 -0.02958518

Non-Significant Differences

Dominant Symbiont by Reef Type

## 
##  Pearson's Chi-squared test with Yates' continuity correction
## 
## data:  results
## X-squared = 0.77593, df = 1, p-value = 0.3784

Dominant Symbiont by Bay Area

## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 1.8678, df = 2, p-value = 0.393

Dominant Symbiont by Reef ID

results=table(Symcap$Dom, Symcap$Reef.ID)
chisq.test(results)
KB <- c(21.46087401, -157.809907) 
KBMap <- GetMap(center = KB, zoom = 13, maptype = "satellite", SCALE = 2, GRAYSCALE = FALSE)
Latitude=aggregate(Latitude~Reef.ID, data=Symcap, FUN = mean)
Longitude=aggregate(Longitude~Reef.ID, data = Symcap, FUN=mean)
XY<-merge(Latitude, Longitude, by="Reef.ID", all=T)
propDom=table(Symcap$Dom, Symcap$Reef.ID)
propDom=prop.table(propDom, margin = 2)
propDom <- as.data.frame.matrix(propDom)
props <- data.frame(t(propDom))
props$Reef.ID <- rownames(props)
XY<-merge(XY, props, by="Reef.ID", all=T)
newcoords <- LatLon2XY.centered(KBMap, XY$Latitude, XY$Longitude, zoom=13)
XY$X <- newcoords$newX
XY$Y <- newcoords$newY
PlotOnStaticMap(KBMap, XY$Latitude, XY$Longitude)

rownames(XY) <- XY$Reef.ID
XY <- XY[, -1]
XY <- na.omit(XY)
apply(XY, MARGIN=1, FUN=function(reef) {
  floating.pie(xpos = reef["X"], ypos = reef["Y"], 
               x=c(reef["C"], reef["D"]), radius = 7, col = c("blue", "red"))
})

Mantel Test for Spatial Autocorrelation
Latitude=aggregate(Latitude~Reef.ID, data=Symcap, FUN = mean)
Longitude=aggregate(Longitude~Reef.ID, data = Symcap, FUN=mean)
XY<-merge(Latitude, Longitude, by="Reef.ID", all=T)
propDom=table(Symcap$Dom, Symcap$Reef.ID)
propDom=prop.table(propDom, margin = 2)
propDom <- as.data.frame.matrix(propDom)
props <- data.frame(t(propDom))
props$Reef.ID <- rownames(props)
XY<-merge(XY, props, by="Reef.ID", all=T)
XY$X <- newcoords$newX
XY$Y <- newcoords$newY
reef.dists <- dist(cbind(XY$Longitude, XY$Latitude))
dom.dists <- dist(XY$C)
mantel(dom.dists~reef.dists)
##    mantelr      pval1      pval2      pval3  llim.2.5% ulim.97.5% 
## 0.15974663 0.04600000 0.95500000 0.05200000 0.09769237 0.24217808

Color Morph by Reef Type

## 
##  Pearson's Chi-squared test with Yates' continuity correction
## 
## data:  results
## X-squared = 1.1302, df = 1, p-value = 0.2877

Color Morph by Bay Area

## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 3.964, df = 2, p-value = 0.1378

Symbiont Community Composition by Reef Type

## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 2.8371, df = 3, p-value = 0.4174

Symbiont Community Composition by Bay Area

## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 8.9692, df = 6, p-value = 0.1753

Symbiont Community Composition by Reef ID

## 
##  Pearson's Chi-squared test
## 
## data:  results
## X-squared = 87.127, df = 72, p-value = 0.1081

Logistic Regression

Significant Effect

Dominant Symbiont by Depth

merged$Dominant <- ifelse(merged$Dom=="C", 0, 1)
Dom1 <- subset(merged, !is.na(newDepth) & !is.na(Dominant))
results=glm(Dominant~newDepth, family = "binomial", data = merged)
anova(results, test = "Chisq")
## Analysis of Deviance Table
## 
## Model: binomial, link: logit
## 
## Response: Dominant
## 
## Terms added sequentially (first to last)
## 
## 
##          Df Deviance Resid. Df Resid. Dev  Pr(>Chi)    
## NULL                       705     942.15              
## newDepth  1   129.04       704     813.10 < 2.2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
logi.hist.plot(Dom1$newDepth, Dom1$Dominant, boxp = FALSE, type = "hist", col="gray", xlabel = "Depth (m)", ylabel = "", ylabel2 = "")
mtext(side = 4, text = "Frequency", line = 3, cex=1)
mtext(side = 4, text = "C                             D", line = 2, cex = 0.75)
mtext(side = 2, text = "Probability of clade C Symbiont", line = 3, cex = 1)

merged$DepthInt <- cut(merged$newDepth, breaks = 0:13)
merged$Dominant2 <- ifelse(merged$Dom=="C", 1, 0)
results=table(merged$Dominant2, merged$DepthInt)
results
##    
##     (0,1] (1,2] (2,3] (3,4] (4,5] (5,6] (6,7] (7,8] (8,9] (9,10] (10,11]
##   0   148    33    28    22    14     4     3     2     2      1       0
##   1    73    52    84    91    47    26    15    12    16      8       5
##    
##     (11,12] (12,13]
##   0       1       0
##   1       0       1
props <- prop.table(results, margin = 2)
par(mar=c(4, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("red", 0.25), alpha("blue", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("C", "D"), fill=c(alpha("blue", 0.25), alpha("red", 0.25)), inset = c(-.2, 0), xpd = NA)
par(new = T)
par(mar=c(4.2, 4, 2, 6))
results=glm(Dominant~newDepth, family = "binomial", data = merged)
fitted <- predict(results, newdata = list(newDepth=seq(0,11,0.1)), type = "response")
plot(fitted~seq(0,11,0.1), xaxs="i", yaxs="i", xlim=c(0,11), ylim=c(0,1), type="l", lwd = 3, xlab="Depth (m)", ylab="Dominant Symbiont Proportion")

Bars indicate relative frequency of clade C vs. D dominance at 1m depth intervals when pooling all samples collected.

Mixture by Depth

merged$Mixture <- ifelse(!merged$Mix=="C" & !merged$Mix=="D", 1, 0)
merged$Mixture2 <- ifelse(!merged$Mix=="C" & !merged$Mix=="D", 0, 1)
results=glm(Mixture~newDepth, family = "binomial", data = merged)
anova(results, test = "Chisq")
## Analysis of Deviance Table
## 
## Model: binomial, link: logit
## 
## Response: Mixture
## 
## Terms added sequentially (first to last)
## 
## 
##          Df Deviance Resid. Df Resid. Dev  Pr(>Chi)    
## NULL                       705     973.27              
## newDepth  1   93.481       704     879.79 < 2.2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
results=table(merged$Mixture2, merged$DepthInt)
results
##    
##     (0,1] (1,2] (2,3] (3,4] (4,5] (5,6] (6,7] (7,8] (8,9] (9,10] (10,11]
##   0   153    44    40    27    18     8     4     2     5      2       1
##   1    68    41    72    86    43    22    14    12    13      7       4
##    
##     (11,12] (12,13]
##   0       1       0
##   1       0       1
props <- prop.table(results, margin = 2)
par(mar=c(4, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("red", 0.25), alpha("blue", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("Mix", "Non-Mix"), fill=c(alpha("blue", 0.25), alpha("red", 0.25)), inset = c(-.23, 0), xpd = NA)
par(new = T)
par(mar=c(4.2, 4, 2, 6))
results=glm(Mixture~newDepth, family = "binomial", data = merged)
fitted <- predict(results, newdata = list(newDepth=seq(0,11,0.1)), type = "response")
plot(fitted~seq(0,11,0.1), xaxs="i", yaxs="i", xlim=c(0,11), ylim=c(0,1), type="l", lwd = 3, xlab="Depth (m)", ylab="Mixture Proportion")

Bars indicate relative frequency of Mixture vs. Non-Mixtures at 1m depth intervals when pooling all samples collected.

Color Morph by Depth

merged$Color <- ifelse(merged$Color.Morph=="Orange", 1, 0)
results=glm(Color~newDepth, family = "binomial", data = merged)
anova(results, test = "Chisq")
## Analysis of Deviance Table
## 
## Model: binomial, link: logit
## 
## Response: Color
## 
## Terms added sequentially (first to last)
## 
## 
##          Df Deviance Resid. Df Resid. Dev  Pr(>Chi)    
## NULL                       706     963.85              
## newDepth  1   78.717       705     885.14 < 2.2e-16 ***
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
Color <- subset(merged, !is.na(newDepth) & !is.na(Color))
logi.hist.plot(independ = Color$newDepth, depend = Color$Color, type = "hist", boxp = FALSE, ylabel = "", col="gray", ylabel2 = "", xlabel = "Depth (m)")
mtext(side = 4, text = "Frequency", line = 3, cex=1)
mtext(side = 4, text = "Brown                       Orange", line = 2, cex = 0.75)
mtext(side = 2, text = "Probability of Orange Color Morph", line = 3, cex = 1)

merged$Color <- ifelse(merged$Color.Morph=="Orange", 0, 1)
results=table(merged$Color, merged$DepthInt)
results
##    
##     (0,1] (1,2] (2,3] (3,4] (4,5] (5,6] (6,7] (7,8] (8,9] (9,10] (10,11]
##   0   171    42    59    61    31    10     9     2     3      1       0
##   1    50    43    53    52    30    20     9    12    16      8       5
##    
##     (11,12] (12,13]
##   0       1       0
##   1       0       1
props <- prop.table(results, margin = 2)
par(mar=c(4, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("orange", 0.25), alpha("sienna", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("Brown", "Orange"), fill=c(alpha("sienna", 0.25), alpha("orange", 0.25)), inset = c(-.22, 0), xpd = NA)
par(new = T)
par(mar=c(4.2, 4, 2, 6))
merged$Color2 <- ifelse(merged$Color=="0", 1, 0)
results=glm(Color2~newDepth, family = "binomial", data = merged)
fitted <- predict(results, newdata = list(newDepth=seq(0,11,0.1)), type = "response")
plot(fitted~seq(0,11,0.1), xaxs="i", yaxs="i", xlim=c(0,11), ylim=c(0,1), type="l", lwd = 3, xlab="Depth (m)", ylab="Color Morph Proportion")

Bars indicate relative frequency of Brown vs. Orange color morph dominance at 1m depth intervals when pooling all samples collected.

Two-Way ANOVA

Significant Interactive Effects

Dominant Symbiont by Color Morph and Reef Area

merged$Reef.Area <- ifelse(merged$Reef.Area!="Top", yes = "Slope", no = "Top")
table(merged$Dom, merged$Color.Morph, merged$Reef.Area)
## , ,  = Slope
## 
##    
##     Brown Orange
##   C   226    122
##   D    21     79
## 
## , ,  = Top
## 
##    
##     Brown Orange
##   C    40     45
##   D    12    161
model=aov(Dominant~Color.Morph*Reef.Area, data = merged)
Anova(model, type = 2)
## Anova Table (Type II tests)
## 
## Response: Dominant
##                        Sum Sq  Df F value    Pr(>F)    
## Color.Morph            21.329   1 134.208 < 2.2e-16 ***
## Reef.Area              14.489   1  91.168 < 2.2e-16 ***
## Color.Morph:Reef.Area   1.780   1  11.201 0.0008612 ***
## Residuals             111.565 702                      
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Dominant Symbiont by Color Morph and Depth

Because an interactive effect of reef area and color morph was observed and slope is indicative of a depth gradient, the interaction between depth and color morph was tested here.

model1=lm(Dominant~Color.Morph*newDepth, data = merged)
Anova(model1, type = 2)
## Anova Table (Type II tests)
## 
## Response: Dominant
##                       Sum Sq  Df F value    Pr(>F)    
## Color.Morph           24.366   1 152.751 < 2.2e-16 ***
## newDepth               9.760   1  61.187 1.909e-14 ***
## Color.Morph:newDepth   6.098   1  38.228 1.068e-09 ***
## Residuals            111.977 702                      
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Depth at which Orange switches from D to C Dominance

While brown was always C-dominated, orange was observed to switch from D to C dominance. The depth threshold at which this switch occurs was analyzed here.

threshdepth <- function(color) {
  df <- subset(merged, Color.Morph==color)
  plot(df$Dominant2~df$newDepth, xlab="Depth (m)", ylab = "Probability of Clade C Symbiont",
       main=color)
  abline(h = 0.5, lty=2)
  results=glm(Dominant2~newDepth, family = "binomial", data = df)
  pval <- data.frame(coef(summary(results)))$`Pr...z..`[2]
  mtext(side=3, text=pval)
  newdata <- list(newDepth=seq(0,11,0.01))
  fitted <- predict(results, newdata = newdata, type = "response")
  lines(fitted ~ seq(0,11,0.01))
  thresh <- ifelse(pval < 0.05,
                   newdata$newDepth[which(diff(sign(fitted - 0.5))!=0)], NA)
  return(thresh)
}
sapply(levels(merged$Color.Morph), FUN=threshdepth)

##  Brown Orange 
##     NA   2.75
df <- subset(merged, Color.Morph=="Orange")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
par(mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="orange", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)
df <- subset(merged, Color.Morph=="Brown")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
fitted <- predict(results, newdata = newdata, type = "response")
lines(fitted~seq(0,11,0.01), col="sienna", lwd=3)
mtext(side = 1, text = "Depth (m)", line = 3, cex = 1)
mtext(side = 2, text = "Probability of Clade C Symbiont", line = 3, cex = 1)
legend("topright", legend=c("Brown", "Orange"), fill=c("sienna", "orange"), inset = c(-.22, 0), xpd = NA)

Dominant Symbiont by Depth and Reef Type

model3=aov(Dominant2~newDepth*Reef.Type, data = merged)
Anova(model3, type = 2)
## Anova Table (Type II tests)
## 
## Response: Dominant2
##                     Sum Sq  Df  F value    Pr(>F)    
## newDepth            24.789   1 123.6146 < 2.2e-16 ***
## Reef.Type            0.016   1   0.0801  0.777311    
## newDepth:Reef.Type   1.650   1   8.2263  0.004253 ** 
## Residuals          140.774 702                       
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
df <- subset(merged, Reef.Type=="Patch")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
par(mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="dodgerblue3", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)
df <- subset(merged, Reef.Type=="Fringe")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
fitted <- predict(results, newdata = newdata, type = "response")
lines(fitted~seq(0,11,0.01), col="brown1", lwd=3)
mtext(side = 1, text = "Depth (m)", line = 3, cex = 1)
mtext(side = 2, text = "Probability of Clade D Symbiont", line = 3, cex = 1)
legend("topright", legend=c("Patch", "Fringe"), fill=c("dodgerblue3", "brown1"), inset = c(-.22, 0), xpd = NA)

Color Morph by Depth and Reef Type

model2=aov(Color~newDepth*Reef.Type, data = merged)
Anova(model2, type = 2)
## Anova Table (Type II tests)
## 
## Response: Color
##                     Sum Sq  Df F value  Pr(>F)    
## newDepth            18.253   1 83.9552 < 2e-16 ***
## Reef.Type            0.069   1  0.3195 0.57208    
## newDepth:Reef.Type   1.289   1  5.9284 0.01515 *  
## Residuals          152.838 703                    
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
df <- subset(merged, Reef.Type=="Patch")
results=glm(Color2~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
par(mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="dodgerblue3", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)
df <- subset(merged, Reef.Type=="Fringe")
results=glm(Color2~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
fitted <- predict(results, newdata = newdata, type = "response")
lines(fitted~seq(0,11,0.01), col="brown1", lwd=3)
mtext(side = 1, text = "Depth (m)", line = 3, cex = 1)
mtext(side = 2, text = "Probability of Orange Color Morph", line = 3, cex = 1)
legend("topright", legend=c("Patch", "Fringe"), fill=c("dodgerblue3", "brown1"), inset = c(-.22, 0), xpd = NA)

Dominant Symbiont per Color Morph by Depth and Reef Type

Because depth and reef type have an interactive effect on both color morph and dominant symbiont clade, the dominant symbiont per color morph at each reef type was visualized.

# Dominant Symbiont per Color Morph by Depth and Reef Type
df <- subset(merged, Reef.Type=="Patch")
dfo <- subset(df, Color.Morph=="Orange")
results=glm(Dominant~newDepth, family = "binomial", data = dfo)
newdata <- list(newDepth=seq(0,11,0.01))
par(mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="dodgerblue3", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)

df <- subset(merged, Reef.Type=="Patch")
dfb <- subset(df, Color.Morph=="Brown")
results=glm(Dominant~newDepth, family = "binomial", data = dfb)
newdata <- list(newDepth=seq(0,11,0.01))
par(new = T, mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="black", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)

df <- subset(merged, Reef.Type=="Fringe")
dfo <- subset(df, Color.Morph=="Orange")
results=glm(Dominant~newDepth, family = "binomial", data = dfo)
newdata <- list(newDepth=seq(0,11,0.01))
par(new = T, mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="red", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)

df <- subset(merged, Reef.Type=="Fringe")
dfb <- subset(df, Color.Morph=="Brown")
results=glm(Dominant~newDepth, family = "binomial", data = dfb)
newdata <- list(newDepth=seq(0,11,0.01))
par(new = T, mar=c(4, 4, 2, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="orange", lwd=3, xlab="", ylab="", axisnames=FALSE)
abline(h = 0.5, lty=2)
legend("topright", legend=c("PO", "PB", "FO", "FB"), fill=c("dodgerblue3", "black", "red", "orange"), inset = c(-.2, 0), xpd = NA)
mtext(side = 2, text = "Probability of Clade D Symbiont", line = 3, cex = 1)
mtext(side = 1, text = "Depth (m)", line = 3, cex = 1)

Dominant Symbiont by Depth and Reef ID

model2=aov(Dominant~newDepth*Reef.Type, data = merged)
Anova(model2, type = 2)
## Anova Table (Type II tests)
## 
## Response: Dominant
##                     Sum Sq  Df  F value    Pr(>F)    
## newDepth            24.789   1 123.6146 < 2.2e-16 ***
## Reef.Type            0.016   1   0.0801  0.777311    
## newDepth:Reef.Type   1.650   1   8.2263  0.004253 ** 
## Residuals          140.774 702                       
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
merged$Dominant2 <- ifelse(merged$Dom=="C", 0, 1)
par(mfrow=c(5,5))

domreef <- function(id) {
  df <- subset(merged, Reef.ID==id)
  results=glm(Dominant2~newDepth, family = "binomial", data = df)
  newdata <- list(newDepth=seq(0,11,0.01))
  par(mar=c(1, 1, 3, 1))
  fitted <- predict(results, newdata = newdata, type = "response")
  plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", 
       col="dodgerblue3", lwd=3, xlab="", ylab="")
  mtext(side = 3, text = id)
  abline(h=0.5, lty=2)
}

sapply(levels(merged$Reef.ID), FUN=domreef)

On the y-axis, a value of 1 indicates D-dominance and a value of 0 indicates C-dominance.

Mixture by Depth and Reef ID

merged$Mixture <- ifelse(!merged$Mix=="C" & !merged$Mix=="D", 1, 0)
model4=aov(Mixture~newDepth*Reef.ID, data = merged)
Anova(model4, type = 2)
## Anova Table (Type II tests)
## 
## Response: Mixture
##                   Sum Sq  Df F value    Pr(>F)    
## newDepth          18.820   1 90.3912 < 2.2e-16 ***
## Reef.ID            9.944  24  1.9900  0.003478 ** 
## newDepth:Reef.ID   7.967  24  1.5944  0.036252 *  
## Residuals        136.581 656                      
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
par(mfrow=c(5,5))

mixreef <- function(id) {
  df <- subset(merged, Reef.ID==id)
  results=glm(Mixture~newDepth, family = "binomial", data = df)
  newdata <- list(newDepth=seq(0,11,0.01))
  par(mar=c(1, 1, 3, 1))
  fitted <- predict(results, newdata = newdata, type = "response")
  plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", 
       col="dodgerblue3", lwd=3, xlab="", ylab="")
  mtext(side = 3, text = id)
  abline(h=0.5, lty=2)
}

sapply(levels(merged$Reef.ID), FUN=mixreef)

On the y-axis, a value of 1 indicates a mixture and a value of 0 indicates single symbiont clade.

Figures

Coral Collection Map

KB <- c(21.46087401, -157.809907) 
KBMap <- GetMap(center = KB, zoom = 13, maptype = "satellite", SCALE = 2, GRAYSCALE = FALSE)
Latitude=aggregate(Latitude~Reef.ID, data=Symcap, FUN = mean)
Longitude=aggregate(Longitude~Reef.ID, data = Symcap, FUN=mean)
XY<-merge(Latitude, Longitude, by="Reef.ID", all=T)
newcoords <- LatLon2XY.centered(KBMap, XY$Latitude, XY$Longitude, zoom=13)
XY$X <- newcoords$newX
XY$Y <- newcoords$newY
XY <- subset(XY, Reef.ID!="37")
par(oma=c(3,3,0,0))
PlotOnStaticMap(KBMap, XY$Latitude, XY$Longitude, col=153, pch=21, bg="#FF7F50", lwd=2)
axis(1, at = LatLon2XY.centered(KBMap, NA, c(-157.85, -157.81, -157.77))$newX, tcl=0.5, line = 0.5, col = "ghostwhite", col.ticks = "black", lwd = 1, outer = TRUE, labels = c("157.85°W", "157.81°W", "157.77°W"), padj = -2.5, cex.axis = 0.75)
axis(2, at = LatLon2XY.centered(KBMap, c(21.42, 21.46, 21.50), NA)$newY, tcl=0.5, line = 0.5, col = "ghostwhite", col.ticks = "black", lwd = 1, outer = TRUE, labels = c("21.42°N", "21.46°N", "21.50°N"), padj = 0.5, hadj = 0.60, las = 1, cex.axis = 0.75)
par(new=T, mar=c(11.8,19,0,0))
HI <- readOGR("coast_n83.shp", "coast_n83") 
HI <- spTransform(HI, CRS("+proj=longlat +datum=NAD83")) 
plot(HI, xlim=c(-158.3, -157.6), ylim=c(21.35, 21.6), lwd=0.4, col="gray", bg="white")
rect(-157.9, 21.41, -157.75, 21.53)
box()

Depth Influence on Dominant Symbiont and Color Morph

par(mfrow=c(3,1))

merged$DepthInt <- cut(merged$newDepth, breaks = 0:13)
merged$Dominant <- ifelse(merged$Dom=="C", 0, 1)
merged$Dominant2 <- ifelse(merged$Dom=="C", 1, 0)
results=table(merged$Dominant2, merged$DepthInt)
results
props <- prop.table(results, margin = 2)
par(mar=c(2, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("red", 0.25), alpha("blue", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("C", "D"), fill=c(alpha("blue", 0.25), alpha("red", 0.25)), inset = c(0, 0), xpd = NA)
par(new = T)
par(mar=c(2.1, 4, 2, 6))
results=glm(Dominant~newDepth, family = "binomial", data = merged)
fitted <- predict(results, newdata = list(newDepth=seq(0,11,0.1)), type = "response")
plot(fitted~seq(0,11,0.1), xaxs="i", yaxs="i", xlim=c(0,11), ylim=c(0,1), type="l", lwd = 3, xlab="", ylab="Probability of D-Dominance")

merged$Color <- ifelse(merged$Color.Morph=="Orange", 0, 1)
results=table(merged$Color, merged$DepthInt)
results
props <- prop.table(results, margin = 2)
par(mar=c(3, 4, 1, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("orange", 0.25), alpha("sienna", 0.25)), 
        xlab = "", ylab = "Probability of Orange-Dominance",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("Brown", "Orange"), fill=c(alpha("sienna", 0.25), alpha("orange", 0.25)), inset = c(0, 0), xpd = NA)
par(new = T)
par(mar=c(3.1, 4, 1, 6))
merged$Color2 <- ifelse(merged$Color=="0", 1, 0)
results=glm(Color2~newDepth, family = "binomial", data = merged)
fitted <- predict(results, newdata = list(newDepth=seq(0,11,0.1)), type = "response")
plot(fitted~seq(0,11,0.1), xaxs="i", yaxs="i", xlim=c(0,11), ylim=c(0,1), type="l", lwd = 3, xlab="", ylab="")

df <- subset(merged, Color.Morph=="Orange")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
par(mar=c(4, 4, 0, 6))
fitted <- predict(results, newdata = newdata, type = "response")
plot(fitted ~ seq(0,11,0.01), ylim = c(0,1), type="l", col="orange", lwd=3, xlab="Depth (m)", ylab="Probabilty of D-Dominance", axisnames=FALSE, xaxs = "i", yaxs = "i")
abline(h = 0.5, lty=2)
df <- subset(merged, Color.Morph=="Brown")
results=glm(Dominant~newDepth, family = "binomial", data = df)
newdata <- list(newDepth=seq(0,11,0.01))
fitted <- predict(results, newdata = newdata, type = "response")
lines(fitted~seq(0, 11, 0.01), col="sienna", lwd=3)
#legend("topright", legend=c("Brown", "Orange"), fill=c("sienna", "orange"), inset = c(-.13, 0), xpd = NA)

results=table(merged$Dominant2, merged$Color.Morph)
chisq.test(results)
prop.table(results, margin = 2)
par(new=T, mar=c(10, 10, .5, 6.3))
barplot(prop.table(results, margin = 2), col = c(alpha("red", 0.35), alpha("blue", 0.35)), xlab = "", ylab = "", yaxt = 'n')

#legend("topright", legend=c("C", "D"), fill=c(alpha("blue", 0.35), alpha("red", 0.35)), inset = c(0, 0), xpd = NA)
merged$DomCol <- interaction(merged$Dom, merged$Color.Morph)
merged$DomCol <- factor(merged$DomCol, levels=rev(levels(merged$DomCol)))
results=table(merged$DomCol, merged$DepthInt)
results
props <- prop.table(results, margin = 2)
par(mar=c(4, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("orange", 0.75), alpha("orange", 0.25), alpha("sienna", 0.75), alpha("sienna", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("OD", "OC", "BD", "BC"), fill=c(alpha("orange", 0.75), alpha("orange", 0.25), alpha("sienna", 0.75), alpha("sienna", 0.25)), inset = c(-.23, 0), xpd = NA)
par(new=T, mar=c(4.2, 4, 2, 6))
box()

merged$DomCol2 <- interaction(merged$Color.Morph, merged$Dom)
merged$DomCol2 <- factor(merged$DomCol2, levels=rev(levels(merged$DomCol2)))
results=table(merged$DomCol2, merged$DepthInt)
results
props <- prop.table(results, margin = 2)
par(mar=c(4, 4, 2, 6), lwd = 0.25)
barplot(props[,1:11], col = c(alpha("orange", 0.75), alpha("sienna", 0.75), alpha("orange", 0.25), alpha("sienna", 0.25)), 
        xlab = "", ylab = "",
        space = 0, xaxs="i", yaxs="i", axisnames = FALSE)
par(lwd=1)
legend("topright", legend=c("OD", "BD", "OC", "BC"), fill=c(alpha("orange", 0.75), alpha("sienna", 0.75), alpha("orange", 0.25), alpha("sienna", 0.25)), inset = c(-.23, 0), xpd = NA)
par(new=T, mar=c(4.2, 4, 2, 6))
box()